Cultures of GFAP nestin cells that differentiate to neurons

ABSTRACT

Cultures of cells immunoreactive for glial fibrillary acidic protein (GFAP), as well as for the intermediate filament marker nestin were grown in a medium including epidermal growth factor (EGF) and serum. The cultured cells had the morphology of astroglial cells. The cells can be proliferated in adherent or suspension cultures. Depending on the culture conditions, the cells can be induced to differentiate to neurons or glial cells. The cultures can be expanded over a large number of passages during several months, and survive, express an astroglial phenotype and integrate well after transplantation into both neonatal and adult rat forebrain.

CLAIM OF PRIORITY

This application is a Continuation of U.S. application Ser. No.09/696,530, filed Oct. 24, 2000, which claims priority to U.S.provisional patent application 60/161,316, filed Oct. 25, 1999, thedisclosures of which are incorporated herein by reference in theirentireties.

TECHNICAL FIELD OF THE INVENTION

This invention relates generally to glial cell cultures, and moreparticularly to methods and media for proliferating and differentiatingGFAP⁺ nestin⁺ cells.

BACKGROUND OF THE INVENTION

The development of the mammalian central nervous system (CNS) begins inthe early stage of embryonic development and continues until thepost-natal period. The first step in neural development is cell birth,which is the precise temporal and spatial sequence in which neural stemcells and neural stem cell progeny (i.e. daughter neural stem cells andneural progenitor cells) proliferate. Proliferating cells give rise toneuroblasts, glioblasts, and new neural stem cells. The next step is aperiod of neural cell differentiation and migration, which give rise tothe neurons and glial cells that migrate to their final positions. Theneural component of the mature mammalian CNS is composed of neuronalcells (neurons) and glial cells (astrocytes and oligodendrocytes).

In mammals, specialized glial cells called radial glia developimmediately before the differentiation and migration of neurons. Theseradial glial cells span the cerebral wall from the ventricular surfaceof the neuroepithelium to the pial surface, forming a scaffolding forthe initiation and maintenance of neuronal cell migration. Through aseries of reciprocal signaling events between the migrating neurons andthe radial glia, neurons migrate from their site of origin to theirfinal position along the elongated processes of the radial glial cells.During neuronal migration, radial glial cells do not divide. Afterneuronal production and migration end, however, the radial glia enter amitotic cycle, eventually differentiating into multipolar astrocytes. Inlower vertebrates, radial glia has the capacity to form neurons but itis currently unclear whether radial glia or other types of glialprecursors have the same capacity in mammals. Collectively, severalstudies suggest that may be only a small (and often reversible)transition between neuroepithelial stem cells and radial glia.

The use of cells for neural transplantation is well documented. Severalstudies have indicated that primary tissue from the developing ventralmesencephalon can give rise to dopaminergic neurons and supporting cellscapable of survival, function, and therapeutic efficacy in Parkinson'spatients. In addition, the transplantation of cultures containing neuralprecursor cells and stem cells can give rise all three major cellsubtypes of the CNS, i.e. neurons, astrocytes, and oligodendrocytes.From these studies, there is a clear need in the art for cells capableof proliferating to make large numbers of cells as well as a capacityfor neural differentiation in order to make the appropriate “adult”cells capable of integrating and restoring function to a diseased areain the CNS. Furthermore, over the past couple of decades, proteinfactors capable of protecting neural cells in the CNS from damage andcapable of restoring function have bee discovered. From neuroprotectionstudies, it is evident that these protein factors may best work ifdelivered by gene manipulated cells placed in the area of disease. Thus,there is also a need in the art for transplantable neural cell linescapable of being gene modified in order to secrete protein factorslocally. In addition, cell lines capable of making neurons and otherneural lineages in a reproducible manner are useful screening targets toidentify factors and drugs capable of influencing the CNS. Hence, thereis a need in the art for neural cell lines for drug screening purposes.Last, with the human genome almost completely sequenced, there is a needfor cells of neural lineages, which can be used to identify cDNAlibraries to screen for gene function.

If glial precursor cells of the mammalian CNS could form neurons,astrocytes and perhaps other subtypes, a dividing pool of glialprecursor cells could become a reliable source of large numbers ofneural cells for the needs described above identifying several areas ofindustrial application. Preferably, cellular division in such glialprecursor cells would be epigenetically regulated, so that a suitablenumber of glial precursor cells could be efficiently prepared insufficient numbers for transplantation. However, these cells could alsobe genetically modified in order to be made to proliferate ordifferentiate in a reproducible manner. Furthermore, the cells could begenetically modified in order to produce a protein factor suitable as atherapeutic. The unmodified or gene modified glial precursor cellsshould be suitable in autografts, xenografts, and allografts as well asfor in vitro use to screen for drug activity or gene expression.Protocols allowing for stable and long-term propagation of glialprecursor cells would therefore be of great value. If such culturescould grow over extended periods, their properties would be interestingto compare to those of neural stem cells.

SUMMARY OF THE INVENTION

The invention provides glial precursor cultures of GFAP⁺ nestin⁺ cellswith the potential to differentiate to neurons or glial cells, dependingon the culture conditions chosen. These cells can be expanded usingproliferation-inducing growth factor. Cells in cultures that have beenexpanded extensively express similar phenotypes to those passaged fewertimes. Alternatively, these cells can be induced to make a significantnumber of neurons when placed under non-proliferating and serum-freeconditions. These neurons show regional characteristics from theirorigin of isolation and will express those markers even after long-termculture. Moreover, these cells can make a significant number ofastrocytes when placed under proliferating serum free conditions.

In one embodiment, cell cultures (for example, mouse or human) can beestablished from cells that have been isolated from the medialganglionic eminence (MGE) and lateral ganglionic eminence (LGE). Thecultured cells display glial morphology and both glial fibrillary acidicprotein (GFAP) and nestin immunoreactivity. In this embodiment, the cellcultures contain cells that express the radial glial marker, RC-2. Thecells are at least bi-potential and can make both non-mitotic GFAP⁺astrocytes or non-mitotic beta-tubulin III⁺ neurons, depending on theculture conditions.

In another embodiment, EGF-stimulated and serum-containing long-termcultures of cells from the embryonic mouse lateral ganglionic eminence(LGE) can be expanded over many passages during several months, survive,express an astroglial phenotype and integrate well after transplantationinto both neonatal and adult rat forebrain. Cells propagated in suchcultures are interesting to compare and contrast with central nervoussystem (CNS) neural stem cells grown as neurospheres in EGF-stimulatedcultures and are useful for studies of astroglial development andmigration, and for use in trials with ex vivo gene transfer.

The invention provides a composition of a GFAP⁺ nestin⁺ cell in aculture medium supplemented with serum and at least oneproliferation-inducing growth factor (for example, epidermal growthfactor (EGF) and/or basic fibroblast growth factor (FGF-2) capable ofundergoing neuronal differentiation.

The invention provides a method for the in vitro proliferation of neuralcells, to produce large numbers of glial precursor cells available fortransplantation that are capable of differentiating into neurons andinto glial cells. The method includes the steps of (a) obtaining neuraltissue from a mammal (e.g., from fetal tissue) (b) dissociating theneural tissue to obtain a cell suspension (c) culturing the cell in aculture medium containing a serum and a proliferation-inducing growthfactor, (d) passaging the proliferated cultured cells. Proliferation andperpetuation of the GFAP⁺ nestin⁺ cells can be carried out either insuspension cultures or by allowing cells to adhere to a fixed substratesuch as tissue coated plastic, polylysine, or laminin.

The invention also provides a method for the in vitro differentiation ofthe proliferated GFAP⁺ nestin⁺ cells to form neurons and glia. Theinvention also provides a method for making regionally specifiedneurons.

The invention provides a method for the in vivo transplantation of GFAP⁺nestin⁺ cells, which includes implanting the GFAP⁺ nestin⁺ cells thathave been proliferated in vitro. Thus, the invention provides a meansfor generating large numbers of undifferentiated and differentiatedneural cells for neurotransplantation into a host to treatneurodegenerative disease, neurological trauma, stroke, or in otherdiseases of the nervous system involving neuronal and glial cell loss orwhere normal function needs to be restored such as in metabolic orstorage diseases. The invention also provides for methods of treatingneurodegenerative disease and neurological trauma.

The invention provides a method for the transfection of GFAP⁺ nestin⁺with vectors which can express the gene products for growth factors,growth factor receptors, and peptide neurotransmitters, or expressenzymes, which are involved in the synthesis of neurotransmitters,including those for amino acids, biogenic amines and neuropeptides, andfor the transplantation of these transfected cells into regions ofneurodegeneration.

The invention provides a method of generating large numbers of neuralcells for screening putative therapeutic agents targeted at the nervoussystem and for models of CNS development, function, and dysfunction. Theinvention also provides a method for the screening of potentialneurologically therapeutic pharmaceuticals using GFAP⁺ nestin⁺ cellsthat have been proliferated in vitro. The invention further provides acDNA library prepared from a GFAP⁺ nestin⁺ cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the differentiation of a GFAP⁺ nestin⁺ cell to anastrocyte (A); the differentiation of a GFAP⁺ nestin⁺ cell to a neuron(B); the proliferation of GFAP⁺ nestin⁺ cel suspension culture asneurospheres (C); or the proliferation of GFAP⁺ nestin⁺ cells inadherent culture (D).

FIG. 2 illustrates, the in vitro and in vivo procedures of theinvention.

FIG. 3 illustrates the generation of neurons from LGE glia.(a)Beta-III-tubulin positive cells were severely deficient in the glialcultures grown in the expansion medium (i.e. containing serum and EGF).Note the lone cell exhibiting a neuronal morphology. (b and c) Threedays after removal of both serum and EGF, numerousbeta-III-tubulin-positive cells with neuronal morphology were present.(d and e) Cultures maintained for 7 days without EGF and serum containedbeta-III-tubulin-positive cells displaying distinctive neuronalmorphologies. (f) High power of an in vitro generated neuron with thepatch-clamp pipette attached. (g) Spike-like activity induced by stepdepolarization (traces on the right; vertical scale, 100 mV; horizontalscale, 500 ms) and spontaneous inward currents (traces on the left;vertical scale 100 pA; horizontal scale, 500 ms) in 5 different cells.

FIG. 4 is a histogram illustrating the astroglial phenotype of theattached cells in vitro, as assessed by GFAP-immunocytochemistry aftereach passage, until passage five, when approximately 90% of the cellswere GFAP⁺.

FIG. 5 are histograms that describe the growth characteristics over therepeated passages, expressed as “Cell doubling time” (hrs) and “Days toconfluency”, after each passage. The cells showed a high growth activityduring the initial four passages, but with a significant decrease ingrowth rate during passages 6-8, after which the rate of divisionincreased again to values similar to those during the initial cultureperiod and then remained stable throughout the test, i.e. at least untilpassage 25 (seven months).

DETAILED DESCRIPTION OF THE INVENTION

GFAP⁺ Nestin⁺ cells. The invention provides “NS4” cells. An NS4 cell isan undifferentiated neural cell that can be induced to proliferate usingthe methods of the present invention. The NS4 cell is capable ofself-maintenance, such that with each cell division, at least onedaughter cell will also be a NS4 cell. A NS4 cell has a glial morphologyand is immunoreactive for both glial fibrillary acidic protein (GFAP)and nestin.

Glial fibrillary acidic protein (GFAP) is an intermediate filamentprotein specifically expressed by astrocytes and glial cells of thecentral nervous system and by Schwann cells, the glial cells of theperipheral nervous system (Jessen et al., 13 J. Neurocytology 923-934(1984) and Fields et al., 8 J. Neuroimmunol. 311-330 (1989)). Anti-GFAPantibodies are commercially available (e.g., a rabbit monoclonalantibody raised against GFAP is available from DAKO).

Nestin is an intermediate filament protein found in many types ofundifferentiated CNS cells. During neurogenesis and gliogenesis, nestinis replaced by cell type-specific intermediate filaments, e.g.neurofilaments and glial fibrillary acidic protein (GFAP). The nestinmarker was characterized by Lendahl et al., 60 Cell 585-595 (1990).Antibodies are available to identify nestin, including the rat antibodyRat401.

The co-expression of GFAP and nestin is compatible with the NS4 cellsbeing a population of astroglial cell precursors. Dividing cellscultured from neonatal rat cerebral cortex, with a typical morphology oftype I astroglial cells, co-express GFAP and nestin (Gallo et al., 15 J.Neurosci 394-406 (1995)). Also reactive astrocytes surrounding anischemic or mechanical lesion site co-express GFAP and nestin (Lin etal., 2 Neurobiol. Dis. 79-85 (1995)). The properties of GFAP⁺ nestin⁺NS4 cells, which can be grown long-term and repeatedly passagedcultures, are also interesting in the light of recent publicationsdescribing the existence of GFAP⁺ cells in the ependymal or subependymalzones (Doetsch et al., 97 Cell 703-16 (1999), Johansson et al., 96 Cell25-34 (1999)) which are the areas containing actively dividing neuralstem and precursor cells.

NS4 cells can be obtained from embryonic neural tissue. The neuraltissue can be obtained from any animal that has neural tissue such asinsects, fish, reptiles, birds, amphibians, mammals and the like. Thepreferred source neural tissue is from mammals, preferably rodents andprimates, and most preferably, mice (see, EXAMPLE 1) and humans (see,EXAMPLE 2).

When NS4 cells are obtained from a heterologous donor, the donor may beeuthanized, and the neural tissue and specific area of interest removedusing a sterile procedure. Areas of interest are any area from which NS4cells can be obtained that can serve to restore function to adegenerated area of the host's nervous system, particularly the host'sCNS. Suitable areas include the medial ganglionic eminence and thelateral ganglionic eminence. Autologous neural tissue can be obtained bybiopsy, or from patients undergoing neurosurgery in which neural tissueis removed, for example, during epilepsy surgery, temporal lobectomiesand hippocampectomies. Human heterologous NS4 cells can be derived fromembryonic or fetal tissue following elective abortion (EXAMPLE 2), orfrom a post-natal, juvenile or adult organ donor.

The present invention provides an alternative way to generate enrichedpopulations of astroglial cells from different regions. The NS4 cellsresemble type I astroglial cells both in vitro and followingimplantation. An interesting migration pattern was observed in theneonatal recipients, with cells migrating along the internal capsuleinto the globus pallidus and some other adjacent structures, whereas,when the grafts were placed into adult recipients, they remained mostlyaround the injection site, with only limited migration into the adjacentstriatum (see, EXAMPLE 7).

While neurons and glia were present in the dissociated cell culture,after several passages, the cultures are severely deficient of cellspossessing neuronal morphologies or expressing neuronal markers. Thesecultures are highly enriched in cells having GFAP and nestinimmunoreactivity and expressing glial morphology. The cell cultures alsocontain cells expressing the radial glial marker RC2.

NS4 cells can be maintained in vitro in long-term cultures. Cells fromthe embryonic mouse lateral ganglionic eminence (LGE) were grown inattached, epidermal growth factor (EGF) stimulated and 10%serum-containing cultures, with around 90% GFAP⁺ nestin⁺ cells, overrepeated passages during several months. After a gradual decline indivision rate during the first 6-8 passages, the cultures thereafterpropagated readily again, with a stable and high growth rate, for atleast seven months. Cells grew as attached GFAP⁺ nestin⁺ cells with anastroglial-like morphology (see EXAMPLE 4).

The cultured mouse NS4 cells were also positive for the mouse-specificneural antibodies M2 (Lagenaur & Schachner, 15 J. Supramol. Struct. CellBiochem. 335-46 (1981)) and M6 (Lagenaur et al., 23 J. Neurobiol. 71-88(1992)). Although M2 has been found to label both glial and neuronalcell surfaces, in cerebellar monolayer cultures and in cerebellar tissuesections (Lagenaur & Schachner, 15 J. Supramol. Struct. Cell Biochem.335-46 (1981)), several in vivo studies have characterized the M2antibody as a reliable marker for astroglial cells (Zhou & Lund, 317 J.Comp. Neurol. 145-55 (1992)). M6 is a neuronal cell surface glycoproteinwith unknown function (Mi et al., 106 Dev. Brain Res. 145-54 (1998);Lagenaur et al., 23 J. Neurobiol. 71-88 (1992); Hankin & Lund, 263 J.Comp. Neurol. 45 5-66 (1987); Wictorin et al., 3 Eur. J. Neurosci.86-101 (1991)). Several in vivo studies have shown that M6 can also beexpressed on glial cells (Mi et al., 106 Dev. Brain Res. 145-54 (1998)).The M6-immunoreactivity is observed in the majority of the cultured NS4cells and therefore does not indicate that the cells have certainneuronal characteristics. The M2 and M6 staining patterns were clearlysimilar to those of GFAP and nestin, with the vast majority of the cellsimmunopositive for all of these four markers, at both early and latepassages.

In addition, some NS4 cells show immunoreactivity for the radial gliamarker RC-2. RC2 is a monoclonal antibody that specifically recognizes aradial glial cell antigen that is expressed at varying amounts duringCNS development. There is a high level of expression during embryonicbrain development, lower levels in early postnatal transitional glia,and none in astrocytes after the second postnatal week. Hunter et al.,33 J. Neurobiol. 459-472 (1997); Mission et al., 44 Dev. Brain Res.95-108 (1988).

NS4 cells were negative when stained for neuronal markers, such as,beta-III tubulin or NeuN. Antibodies recognizing-, beta-tubulin isotypeIII (beta-III-tubulin) are commercially available (for example, mousemonoclonal antibodies from Sigma Chemicals, St. Louis Mo.). Antibody toNeuro-Specific Nuclear Protein (NeuN) reacts with most neuronal celltypes throughout the nervous system, is available from Chemicon(Temecula Calif.). The antibody is neuron-specific; no staining of gliais observed. Other neuronal markers include the homeobox-related murinegene MEIS2 labels the lateral somitic compartment and derivatives duringearly mouse embryogenesis and later becomes a marker for thedorso-ectodermal region, overlying cells of the paraxial mesoderm. MEIS2is also highly expressed in specific areas of the developing centralnervous system from embryonic day 9 (E9) onward. In later developmentalstages, a strong expression is detectable in differentiating nuclei andregions of the forebrain, midbrain, hindbrain, and spinal cord. (see,Toresson et al., 126 (6) Development 1317-1326 (1999)). Another neuronalmarker is the DLX homeobox gene, which is expressed in distinct regionsof the embryonic forebrain, including the striatum, neocortex and retina(see, Eisenstat et al., 414(2) J. Comp. Neurol. 217-37 (1999))

NS4 cells survive transplantation into neonatal or adult animalstriatum, with astroglia-like properties for the implanted cells, andgood integration and migration, especially in the neonatal recipients.Transplantation of astroglial cells is today a widely used method for invivo studies of astroglial cells during development and in regeneration,often with the cells grafted as a component of primary tissue, with amix of different precursor cells. To determine specific properties ofthe astroglial cells, it is however interesting to be able to acquirerelatively pure cell populations.

Contrast between NS4 cells of the invention and CNS neural stem cells.The NS4 cells of the invention are similar to and yet different from CNSneural stem cells. Neurobiologists have used various termsinterchangeably to describe the undifferentiated cells of the CNS. Termssuch as “stem cell”, “precursor cell” and “progenitor cell” are commonlyused in the scientific literature to describe different types ofundifferentiated neural cells, with differing characteristics and fates.One approach to obtain CNS neural stem cells is to trophicfactor-stimulate and grow neural stem (or progenitor) cells in the formof neurospheres (see, U.S. Pat. Nos. 5,750,376 and 5,851,832, to Weisset al. U.S. Pat. No. 5,753,506, to Johe, U.S. Pat. No. 5,968,829, toCarpenter (all incorporated herein by reference), Weiss et al., 19Trends Neurosci. 387-93 (1996); Reynolds et al., 12 J. Neurosci. 4565-74(1992), Reynolds & Weiss, 255 Science 1707-10 (1992); Reynolds & Weiss,175 Dev. Biol. 1-13 (1996)). Indeed, such precursor cells derived from,for instance, the embryonic or adult rodent or human forebrain, can begrown and multiplied as non-attached neurospheres over long periods, ina serum-free medium including EGF. In vitro, cells in the neurospheresdifferentiate into neurons, astrocytes or oligodendrocytes, when platedonto an adhesive substrate.

Cells isolated from the embryonic (or adult) mouse striatum canproliferate in response to EGF-stimulation and grow in a medium withoutserum, as non-attached clusters or spheres of clonally derivedundifferentiated progenitor/stem cells, but with a potential todifferentiate into neurons, astrocytes or oligodendrocytes. EGF-expandedneurospheres are nestin⁺ but GFAP⁻. Upon transplantation into the CNS,the neurospheres give rise to neuronal, glial, and nonneural cells andare capable of differentiating into various neurons such as hippocampalneurons of the granule cell layer and olfactory interneurons (Hammang etal., 147 Exp. Neurol. 84-95 (1997); Winkler et al., 11 Mol. CellNeurosci. 99-116 (1998)); Fricker et al., 19 J. Neurosci. 5990-6005(1999), Svendsen et al., 148 Exp. Neurol. 135-46 (1997), and Vescovi etal., 156 Exp. Neurol. 71-83 (1999)). NS4 cells appear to be morerestricted and retain their regional specification and, for example,cells differentiated from precursors derived from the LGE and propagatedfor multiple passages expressed striatal neuronal markers such as MEIS2and DLX1 and did not express markers of cortical or medial ganglioniceminence neuronal precursors (see EXAMPLE 5).

Culture conditions. NS4 cells can be proliferated using the methodsdescribed herein. Cells can be obtained from donor tissue bydissociation of individual cells from the connecting extracellularmatrix of the tissue (see, EXAMPLES 1-2). Tissue from a particularneural region is removed from the brain using a sterile procedure, andthe cells are dissociated using any method known in the art includingtreatment with enzymes such as trypsin, collagenase and the like, or byusing physical methods of dissociation such as with a blunt instrumentor homogenizer. Dissociation of fetal cells can be carried out in tissueculture medium. Dissociation of juvenile and adult cells can be carriedout in 0.1% trypsin and 0.05% DNase in DMEM. Dissociated cells arecentrifuged at low speed, between 200 and 2000 rpm, usually between 400and 800 rpm, and then resuspended in a culture medium. The neural cellscan be cultured in suspension or on a fixed substrate. Dissociated cellsuspensions are seeded in any receptacle capable of sustaining cells,particularly culture flasks, culture plates or roller bottles, and moreparticularly in small culture flasks such as 25 cm² culture flasks.Cells cultured in suspension are resuspended at approximately 5×10⁴ to2×10⁵ cells/ml (for example, 1×10⁵ cells/ml). Cells plated on a fixedsubstrate are plated at approximately 2-3×10³ 10 cells/cm² (for example,2.5×10³ cells/cm²).

The dissociated neural cells can be placed into any known culture mediumcapable of supporting cell growth, including HEM, DMEM, RPMI, F-12, andthe like, containing supplements which are required for cellularmetabolism such as glutamine and other amino acids, vitamins, mineralsand proteins such as transferrin and the like. The culture medium mayalso contain antibiotics to prevent contamination with yeast, bacteriaand fungi such as penicillin, streptomycin, gentamicin and the like. Theculture medium may contain serum derived from bovine, equine, chickenand the like.

In one embodiment, the invention provides a culture medium for theproliferation of NS4 cells. The medium is a defined culture mediumcontaining a mixture of DMEM/F12, supplemented with N2 (Gibco), andfetal calf serum. This culture medium is referred to as “NS4 CompleteMedium” and is described in detail in EXAMPLE 1.

Conditions for culturing should be close to physiological conditions.The pH of the culture medium should be close to physiological pH. (forexample, between pH 6-8, between about pH 7 to 7.8, or at pH 7.4).Physiological temperatures range between about 30° C. to 40° C. NS4cells can be cultured at temperatures between about 32° C. to about 38°C. (for example, between about 35° C. to about 37° C.).

The culture medium is supplemented with at least oneproliferation-inducing (“mitogenic”) growth factor. A “growth factor” isprotein, peptide or other molecule having a growth,proliferation-inducing, differentiation-inducing, or trophic effect onNS4 cells. “Proliferation-inducing growth factors” are trophic factorthat allows NS4 cells to proliferate, including any molecule that bindsto a receptor on the surface of the cell to exert a trophic, orgrowth-inducing effect on the cell. Proliferation-inducing growthfactors include EGF, amphiregulin, acidic fibroblast growth factor (aFGFor FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforminggrowth factor alpha (TGFα), and combinations thereof. EGF is a knownproliferation-inducing growth factor for astroglial cells (Simpson etal., 8 J. Neurosci. Res. 453-62 (1982)), and is also used in media forthe propagation of CNS neural stem cells (Reynolds & Weiss, 255 Science1707-10 (1992)). The combination of the EGF-containing neurospheregrowth medium with the addition of serum gives rise to readilypropagating attached cultures with high proportions of GFAP⁺ cells.

Growth factors are usually added to the culture medium at concentrationsranging between about 1 fg/ml to 1 mg/ml. Concentrations between about 1to 100 ng/ml are usually sufficient. Simple titration assays can easilybe performed to determine the optimal concentration of a particulargrowth factor.

The biological effects of growth and trophic factors are generallymediated through binding to cell surface receptors. The receptors for anumber of these factors have been identified and antibodies andmolecular probes for specific receptors are available. NS4 cells can beanalyzed for the presence of growth factor receptors at all stages ofdifferentiation. In many cases, the identification of a particularreceptor provides guidance for the strategy to use in furtherdifferentiating the cells along specific developmental pathways with theaddition of exogenous growth or trophic factors.

Generally, after about 3-10 days in vitro, and more particularly afterabout 6-7 days in vitro, the proliferating NS4 cells are fed every 2-7days (for example, every 2-4 days by aspirating the medium, and addingfresh “NS4 Complete Medium” containing a proliferation-inducing growthfactor to the culture flask).

The NS4 cell culture can be easily passaged to reinitiate proliferation.After 6-7 days in vitro, the culture flasks are shaken well and NS4cells The NS4 cells are then transferred to a 50 ml centrifuge tube andcentrifuged at low speed. The medium is aspirated, the NS4 cells areresuspended in a small amount of “NS4 Complete Medium” with growthfactor The cells are then counted and replated at the desired density toreinitiate proliferation. This procedure can be repeated weekly toresult in a logarithmic increase in the number of viable cells at eachpassage. The procedure is continued until the desired number of NS4cells is obtained.

NS4 cells and NS4 cell progeny can be cryopreserved by any method knownin the art until they are needed. (See, e.g., U.S. Pat. No. 5,071,741,PCT International patent applications WO†93/14191, WO†95/07611,WO†96/27287, WO†96/29862, and WO†98/14058, Karlsson et al., 65Biophysical J. 2524-2536 (1993)). The NS4 cells can be suspended in anisotonic solution, preferably a cell culture medium, containing aparticular cryopreservant. Such cryopreservants include dimethylsulfoxide (DMSO), glycerol and the like. These cryopreservants are usedat a concentration of 5-15% (for example, 8-10%). Cells are frozengradually to a temperature of −10° C. to −150° C. (for example, −20° C.to −100° C., or −70° C. to −80° C.)

Differentiation of NS4 Cells. Depending on the culture conditions, NS4cells can be differentiated into neurons and glial cells.

NS4 cells can be differentiated into neurons by culturing the NS4 cellson a fixed substrate in a culture medium that is free of theproliferation-inducing growth factor and serum. After removal of theproliferation-inducing growth factor and the serum, the NS4 cells beginto differentiate into neurons. At this stage the culture medium maycontain serum such as 0.5-1.0% fetal bovine serum (FBS). However, ifdefined conditions are required, serum is not used. Within 2-3 days,many of the NS4 cell progeny begin to lose immunoreactivity for GFAP andnestin and begin to express antigens specific for neurons(e.g.,β-tubulin III). Under the same conditions, NS4 cells can bedifferentiated into mature astrocytes by culturing the cells on a fixedsubstrate in a culture medium that is free or deficient of serum. Afterremoval of the serum, the cells flatten, and begin to differentiate intoglia. Cells exhibit the astroglial morphology and lose immunoreactivityfor nestin and begin to express GFAP in a fibrillary patterncharacteristic for astrocytes.

Differentiation of the NS4 cells can also be induced by any method knownin the art which activates the cascade of biological events which leadto growth, which include the liberation of inositol triphosphate andintracellular Ca²⁺, liberation of diacyl glycerol and the activation ofprotein kinase C and other cellular kinases, and the like. Treatmentwith phorbol esters, differentiation-inducing growth factors and otherchemical signals can induce differentiation. Instead ofproliferation-inducing growth factors for the proliferation of NS4 cells(see above), differentiation-inducing growth factors can be added to theculture medium to influence differentiation of the NS4 cells.Differentiation inducing growth factors include NGF, platelet-derivedgrowth factor (PDGF), thyrotropin releasing hormone (TRH), transforminggrowth factor betas (TGF), insulin-like growth factor (IGF-1) and thelike.

Differentiated neuronal and glia cells can be detected usingimmunocytochemical techniques know in the art. Immunocytochemistry (e.g.dual-label immunofluorescence and immunoperoxidase methods) usesantibodies that detect cell proteins to distinguish the cellularcharacteristics or phenotypic properties of neurons from glia. Cellularmarkers for neurons include NSE, NF, β-tubulin, MAP-2 and NeuN. Cellularmarkers for glia include GFAP (an identifier of astrocytes), RC-2 (anidentifier of radial glia) and M2.

Immunocytochemistry can also be used to identify neurons, by detectingthe expression of neurotransmitters or the expression of enzymesresponsible for neurotransmitter synthesis. For the identification ofneurons, antibodies can be used that detect the presence ofacetylcholine (ACh), dopamine, epinephrine, norepinephrine, histamine,serotonin or 5-hydroxytryptamine (5-HT), neuropeptides such as substanceP, adrenocorticotrophic hormone, vasopressin or anti-diuretic hormone,oxytocin, somatostatin, angiotensin II, neurotensin, and bombesin,hypothalamic releasing hormones such as TRH and luteinizing releasinghormone, gastrointestinal peptides such as vasoactive intestinal peptide(VIP) and cholecystokinin (CCK) and CCK-like peptide, opioid peptidessuch as endorphins and enkephalins, prostaglandins, amino acids such asGABA, glycine, glutamate, cysteine, taurine and aspartate, anddipeptides such as carnosine. Antibodies toneurotransmitter-synthesizing enzymes can also be used such as glutamicacid decarboxylase (GAD) which is involved in the synthesis of GABA,choline acetyltransferase (ChAT) for ACh synthesis, dopa decarboxylase(DDC) for dopamine, dopamine-β-hydroxylase (DBH) for norepinephrine, andamino acid decarboxylase for 5-HT. Antibodies to enzymes that areinvolved in the deactivation of neurotransmitters may also be usefulsuch as acetyl cholinesterase (AChE) which deactivates ACh. Antibodiesto enzymes involved in the reuptake of neurotransmitters into neuronalterminals such as monoamine oxidase and catechol-o-methyl transferasefor dopamine, for 5-HT, and GABA transferase for GABA may also identifyneurons. Other markers for neurons include antibodies toneurotransmitter receptors such as the AChE nicotinic and muscarinicreceptors, adrenergic receptors, the dopamine receptor, and the like.Cells that contain a high level of melanin, such as those found in thesubstantia nigra, could be identified using an antibody to melanin.

In situ hybridization histochemistry can also be performed, using cDNAor RNA probes specific for the peptide neurotransmitter or theneurotransmitter synthesizing enzyme mRNAs. These techniques can becombined with immunocytochemical methods to enhance the identificationof specific phenotypes. If necessary, the antibodies and molecularprobes discussed above can be applied to Western and Northern blotprocedures respectively to aid in cell identification.

Transplantation of NS4 Cells. Transplantation of new cells into thedamaged CNS has the potential to repair damaged neural pathways andprovide neurotransmitters, thereby restoring neurological function.However, the absence of suitable cells for transplantation purposes hasprevented the full potential of this procedure from being met.“Suitable” cells are cells that meet the following criteria: (1) can beobtained in large numbers; (2) can be proliferated in vitro to allowinsertion of genetic material, if necessary; (3) capable of survivingindefinitely but stop growing after transplantation to the brain; (4)are non-immunogenic, preferably obtained from a patient's own tissue orfrom a compatible donor; (5) are able to form normal neural connectionsand respond to neural physiological signals (Björklund, 14(8) TrendsNeurosci. 319-322 (1991). The NS4 cells obtainable from embryonic oradult CNS tissue, which are able to divide over extended times whenmaintained in vitro using the culture conditions described herein, meetall of the desirable requirements of cells suitable for neuraltransplantation purposes and are a particularly suitable cell line asthe cells have not been immortalized and are not of tumorigenic origin.The use of NS4 cells in the treatment of neurological disorders and CNSdamage can be demonstrated by the use of animal models.

NS4 cells can be administered to any animal with abnormal neurologicalor neurodegenerative symptoms obtained in any manner, including thoseobtained as a result of mechanical, chemical, or electrolytic lesions,as a result of aspiration of neural areas, or as a result of agingprocesses. Lesions in non-human animal models can be obtained with6-hydroxy-dopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP), ibotenic acid, and the like.

NS4 cells can be prepared from donor tissue that is xenogeneic to thehost. For xenografts to be successful, some method of reducing oreliminating the immune response to the implanted tissue is usuallyemployed. Thus NS4 cell recipients can be immunosuppressed, eitherthrough the use of immunosuppressive drugs such as cyclosporin, orthrough local immunosuppression strategies employing locally appliedimmunosuppressants. Local immunosuppression is disclosed by Gruber, 54Transplantation 1-11(1992). U.S. Pat. No. 5,026,365 disclosesencapsulation methods suitable for local immunosuppression.

As an alternative to employing immunosuppression techniques, methods ofgene replacement or knockout using homologous recombination in embryonicstem cells, taught by Smithies et al., 317 Nature 230-234 (1985), andextended to gene replacement or knockout in cell lines (Zheng et al., 88Proc. Natl. Acad. Sci. 8067-8071 (1991)), can be applied to NS4 cellsfor the ablation of major histocompatibility complex (MHC) genes. NS4cells lacking MHC expression allows for the grafting of enriched neuralcell populations across allogeneic, and perhaps even xenogeneic,histocompatibility barriers without the need to immunosuppress therecipient. General reviews and citations for the use of recombinantmethods to reduce antigenicity of donor cells are also disclosed byGruber, 54 Transplantation 1-11(1992). Exemplary approaches to thereduction of immunogenicity of transplants by surface modification aredisclosed by PCT International patent application WO 92/04033 andPCT/US99/24630. Alternatively the immunogenicity of the graft may bereduced by preparing NS4 cells from a transgenic animal that has alteredor deleted MHC antigens.

NS4 cells can be encapsulated and used to deliver factors to the host,according to known encapsulation technologies, includingmicroencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and5,084,350, herein incorporated by reference) and macroencapsulation(see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881, 4,976,859 and 4,968,733and PCT International patent applications WO 92/19195 and WO 95/05452,each incorporated herein by reference). If the cells are encapsulated,we prefer macroencapsulation, as described in U.S. Pat. Nos. 5,284,761;5,158,881; 4,976,859; 4,968,733; 5,800,828 and PCT International patentapplication WO 95/05452, each incorporated herein by reference. Cellnumber in the devices can be varied; preferably each device containsbetween 10³-10⁹ cells (for example, 10⁵ to 10⁷ cells). Manymacroencapsulation devices can be implanted in the host; we preferbetween one to 10 devices.

NS4 cells prepared from tissue that is allogeneic to that of therecipient can be tested for use by the well-known methods of tissuetyping, to closely match the histocompatibility type of the recipient.

NS4 cells can sometimes be prepared from the recipient's own nervoussystem (e.g., in the case of tumor removal biopsies). In such instancesthe NS4 cells can be generated from dissociated tissue and proliferatedin vitro using the methods described above. Upon suitable expansion ofcell numbers, the NS4 cells may be harvested, genetically modified ifnecessary, and readied for direct injection into the recipient's CNS.

Transplantation can be done bilaterally, or, in the case of a patientsuffering from Parkinson's Disease, contralateral to the most affectedside. Surgery is performed in a manner in which particular brain regionsmay be located, such as in relation to skull sutures, particularly witha stereotaxic guide. NS4 cells are delivered throughout any affectedneural area, in particular to the basal ganglia, the caudate, theputamen, the nucleus basalis or the substantia nigra. Cells areadministered to the particular region using any method which maintainsthe integrity of surrounding areas of the brain, such as by injectioncannula. Injection methods are exemplified by those used by Duncan etal., 17 J. Neurocytology 351-361 (1988), and scaled up and modified foruse in humans. Methods taught by Gage et al., supra, for the injectionof cell suspensions such as fibroblasts into the CNS can also be usedfor injection of NS4 cells. Additional approaches and methods may befound in Neural Grafting in the Mammalian CNS, Björklund & Stenevi, eds.(1985).

NS4 cells administered to the particular neural region can form a neuralgraft, so that the cells form normal connections with neighboringneurons, maintaining contact with transplanted or existing glial cells,and providing a trophic influence for the neurons. Thus the transplantedNS4 cells re-establish the neuronal networks which have been damaged dueto disease and aging.

Survival of the NS4 cell graft in the living host can be examined usingvarious non-invasive scans such as computerized axial tomography (CATscan or CT scan), nuclear magnetic resonance or magnetic resonanceimaging (NMR or MRI), or positron emission tomography (PET) scans.Post-mortem examination of graft survival can be done by removing theneural tissue, and examining the affected region macroscopically andmicroscopically. Cells can be stained with any stains visible underlight or electron microscopic conditions, more particularly with stainsthat are specific for neurons and glia. Particularly useful aremonoclonal antibodies that identify neuronal cell surface markers suchas the M6 antibody that identifies mouse neurons. Also useful areantibodies that identify neurotransmitters (such as GABA, TH, ChAT, andsubstance P) and to enzymes involved in the synthesis ofneurotransmitters (such as GAD). Transplanted cells can also beidentified by prior incorporation of tracer dyes such asrhodamine-labeled or fluorescein-labeled microspheres, fast blue,bisbenzamide, or retrovirally introduced histochemical markers such asthe lacZ gene, which produces, α-galactosidase.

Functional integration of the graft into the host's neural tissue can beassessed by examining the effectiveness of grafts on restoring variousfunctions, including but not limited to tests for endocrine, motor,cognitive and sensory functions. Motor tests that can be used includethose that measure rotational movement away from the degenerated side ofthe brain, and those that measure slowness of movement, balance,coordination, akinesia or lack of movement, rigidity and tremors.Cognitive tests include various tests of ability to perform everydaytasks, as well as various memory tests, including maze performance.

The ability to expand NS4 cells in vitro for use in transplantation isalso useful for ex vivo gene therapy. For instance, rat primaryastroglial cells (Lundberg et al., 139 Exp. Neurol. 39-53 (1996) or ahuman astroglial cell line (Tornatore et al., 5 Cell Transplant 145-63(1996)) have been transduced with the tyrosine hydroxylase gene andimplanted in models of Parkinsonis disease. More recently, astroglialcells for ex vivo gene therapy have also been derived from adult humancortex (Ridet et al., 10 Hum. Gene Ther. 27 1-80 (1999)). Thus, NS4cells provide an additional way to retrieve and expand astroglial cellsfor use as vehicles in ex vivo gene therapy trials.

Genetic Modification of NS4 Cells. Although the NS4 cells arenon-transformed primary cells, they possess features of a continuouscell line. In the undifferentiated state, the NS4 cells continuouslydivide and are thus targets for genetic modification. In someembodiments, the genetically modified cells are induced to differentiateinto neurons or glia by any of the methods described above.

The term “genetic modification” refers to the stable or transientalteration of the genotype of a NS4 cell by intentional introduction ofexogenous DNA. DNA may be synthetic, or naturally derived, and maycontain genes, portions of genes, or other useful DNA sequences. Theterm “genetic modification” as used herein is not meant to includenaturally occurring alterations such as that which occurs throughnatural viral activity, natural genetic recombination, or the like.

Any useful genetic modification of the cells is within the scope of thepresent invention. For example, NS4 cells may be modified to produce orincrease production of a biologically active substance such as aneurotransmitter or growth factor or the like. In one embodiment the thebiologically active substance is a transcription factor such as atranscription factor that modulates genetic differentiation, e.g.,Nurr-1. In an alternative embodiment the biologically active substanceis a non-mitogenic proliferation factor, e.g. v-myc, SV-40 large T ortelomerase.

The genetic modification can be performed either by infection with viralvectors (retrovirus, modified herpes viral, herpes-viral, adenovirus,adeno-associated virus, and the like) or transfection using methodsknown in the art (lipofection, calcium phosphate transfection,DEAE-dextran, electroporation, and the like) (see, Maniatis et al., inMolecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory,N.Y., 1982)). For example, the chimeric gene constructs can containviral, for example retroviral long terminal repeat (LTR), simian virus40 (SV40), cytomegalovirus (CMV); or mammalian cell-specific promoterssuch as tyrosine hydroxylase (TH, a marker for dopamine cells), DBH,phenylethanolamine N-methyltransferase (PNMT), CHAT, GFAP, NSE, the NFproteins (NE-L, NF-M, NF-H, and the like) that direct the expression ofthe structural genes encoding the desired protein. In addition, thevectors can include a drug selection marker, such as the E. coliaminoglycoside phosphotransferase gene, which when co-infected with thetest gene confers resistance to geneticin (G418), a protein synthesisinhibitor.

NS4 cells can be genetically modified using transfection with expressionvectors. In one protocol, vector DNA containing the genes are diluted in0.1×TE (1 mM Tris pH 8.0, 0.1 mM EDTA) to a concentration of 40 μg/ml.22 μl of the DNA is added to 250 μl of 2×HBS (280 mM NaCl, 10 mM KCl,1.5 mM Na₂HPO₄, 12 mM dextrose, 50 mM HEPES) in a disposable, sterile 5ml plastic tube. 31 μl of 2 M CaCl₂ is added slowly and the mixture isincubated for 30 minutes (min) at room temperature. During this 30 minincubation, the cells are centrifuged at 800 g for 5 min at 4° C. Thecells are resuspended in 20 volumes of ice-cold PBS and divided intoaliquots of 1×10⁷ cells, which are again centrifuged. Each aliquot ofcells is resuspended in 1 ml of the DNA-CaCl₂ suspension, and incubatedfor 20 min at room temperature. The cells are then diluted in growthmedium and incubated for 6-24 hr at 37° C. in 5%-7% CO₂. The cells areagain centrifuged, washed in PBS and returned to 10 ml of growth mediumfor 48 hr.

NS4 cells can also be genetically modified using calcium phosphatetransfection techniques. For standard calcium phosphate transfection,the cells are mechanically dissociated into a single cell suspension andplated on tissue culture-treated dishes at 50% confluence (50,000-75,000cells/cm²) and allowed to attach overnight. In one protocol, themodified calcium phosphate transfection procedure is performed asfollows: DNA (15-25 μg) in sterile TE buffer (10 mM Tris, 0.25 mM EDTA,pH 7.5) diluted to 440 μL with TE, and 60 μL of 2 M CaCl² (pH to 5.8with 1M HEPES buffer) is added to the DNA/TE buffer. A total of 500 μLof 2×HBS (HEPES-Buffered saline; 275 mM NaCl, 10 mM KCl, 1.4 mM Na₂HPO_(4,) 12 mM dextrose, 40 mM HEPES buffer powder, pH 6.92) is addeddropwise to this mix. The mixture is allowed to stand at roomtemperature for 20 mm. The cells are washed briefly with 1×HBS and 1 mlof the calcium phosphate precipitated DNA solution is added to eachplate, and the cells are incubated at 37° C. for 20 min. Following thisincubation, 10 ml of “NS4 Complete Medium” is added to the cells, andthe plates are placed in an incubator (37° C., 9.5% CO₂) for anadditional 3-6 hours. The DNA and the medium are removed by aspirationat the end of the incubation period, and the cells are washed 3 timeswith “NS4 Complete Growth Medium” and then returned to the incubator.

When the genetic modification is for the production of a biologicallyactive substance, the substance can be one that is useful for thetreatment of a given CNS disorder. NS4 cells can be genetically modifiedto express a biologically active agent, such as growth factors, growthfactor receptors, neurotransmitters, neurotransmitter synthesizinggenes, neuropeptides, and chromaffin granule amine transporter. Forexample, it may be desired to genetically modify cells so they secrete aproliferation-inducing growth factor or a differentiation-inducinggrowth factor. Growth factor products useful in the treatment of CNSdisorders include NGF, BDNF, the neurotrophins, CNTF, amphiregulin,FGF-1, FGF-2, EGF, TGFα, TGF, PDGF, IGFs, and the interleukins.

Cells can also be modified to express a certain growth factor receptor(r) including, but not limited to, p75 low affinity NGF receptor, CNTFreceptor, the trk family of neurotrophin receptors (trk, trkB, trkC),EGFr, FGFr, and amphiregulin receptors. Cells can be engineered toproduce various neurotransmitters or their receptors such as serotonin,L-dopa, dopamine, norepinephrine, epinephrine, tachykinin, substance P,endorphin, enkephalin, histamine, N-methyl D-aspartate, glycine,glutamate, GABA, ACh, and the like. Useful neurotransmitter-synthesizinggenes include TH, DDC, DBH, PNMT, GAD, tryptophan hydroxylase, ChAT, andhistidine decarboxylase. Genes that encode for various neuropeptides,which may prove useful in the treatment of CNS disorders, includesubstance-P, neuropeptide-Y, enkephalin, vasopressin, VIP, glucagon,bombesin, CCK, somatostatin, calcitonin gene-related peptide, and thelike.

The genetically modified NS4 cells can be implanted for cell therapy orgene therapy into the CNS of a recipient in need of the biologicallyactive molecule produced by the genetically modified cells.Transplantation techniques are detailed below.

Alternatively, the genetically modified NS4 cell can be subjected tovarious differentiation protocols in vitro prior to implantation. Forexample, genetically modified NS4 cells may be removed from the culturemedium, which allows proliferation and differentiated using any of theprotocols, described above. The protocol used depends upon the type ofgenetically modified cell desired. Once the cells have differentiated,they are again assayed for expression of the desired protein. Cellshaving the desired phenotype can be isolated and implanted intorecipients in need of the protein or biologically active molecule thatis expressed by the genetically modified cell.

Methods for screening effects of drugs on NS4 cells. NS4 cell culturescan be used for the screening of potential neurologically therapeuticcompositions. These test compositions can be applied to cells in cultureat varying dosages, and the response of the cells monitored for varioustime periods. Physical characteristics of the cells can be analyzed byobserving cell and neurite growth with microscopy. The induction ofexpression of new or increased levels of proteins such as enzymes,receptors and other cell surface molecules, or of neurotransmitters,amino acids, neuropeptides and biogenic amines can be analyzed with anytechnique known in the art which can identify the alteration of thelevel of such molecules. These techniques include immunohistochemistryusing antibodies against such molecules, or biochemical analysis. Suchbiochemical analysis includes protein assays, enzymatic assays, receptorbinding assays, enzyme-linked immunosorbant assays (ELISA),electrophoretic analysis, analysis with high performance liquidchromatography (HPLC), Western blots, and radioimmune assays (RIA).Nucleic acid analysis such as Northern blots can be used to examine thelevels of mRNA coding for these molecules, or for enzymes whichsynthesize these molecules.

Alternatively, NS4 cells treated with these pharmaceutical compositionscan be transplanted into an animal, and their survival, their ability toform neural connections, and their biochemical and immunologicalcharacteristics examined.

NS4 cells can be used in methods of determining the effect of abiological agents on neural cells. The term “biological agent” refers toany agent, such as a virus, protein, peptide, amino acid, lipid,carbohydrate, nucleic acid, nucleotide, drug, pro-drug or othersubstance that may have an effect on neural cells whether such effect isharmful, beneficial, or otherwise. Biological agents that are beneficialto neural cells are referred to herein as “neurological agents”, a termwhich encompasses any biologically or pharmaceutically active substancethat may prove potentially useful for the proliferation, differentiationor functioning of CNS cells or treatment of neurological disease ordisorder. For example, the term may encompass certain neurotransmitters,neurotransmitter receptors, growth factors, growth factor receptors, andthe like, as well as enzymes used in the synthesis of these agents.

The biological agent can be the biological agent is selected from thegroup consisting of basic fibroblast growth factor, acid fibroblastgrowth factor, epidermal growth factor, transforming growth factor α,transforming growth factor β, nerve growth factor, insulin like growthfactor, platelet derived growth factor, glia-derived neurotrophicfactor, brain derived neurotrophic factor, ciliary neurotrophic factor,phorbol 12-myristate 13-acetate, tryophotin, activin, thyrotropinreleasing hormone, interleukins, bone morphogenic protein, macrophageinflammatory proteins, heparan sulfate, amphiregulin, retinoic acid,tumor necrosis factor α, fibroblast growth factor receptor, epidermalgrowth factor receptor. Examples of biological agents include trophicfactors such as glial-derived neurotrophic factor (GDNF); regulators ofintracellular pathways associated with growth factor activity such asstaurosporine, CGP-4 1251, and the like; hormones; various proteins andpolypeptides such as interleukins and the Bcl1-2 gene product;oligonucleotides such as antisense strands directed, for example,against transcripts for receptors; heparin-like molecules; and a varietyof other molecules that have an effect on radial glial cells or CNSneural stem cell.

To determine the effect of a potential biological agent on neural cellsfrom a particular host, a culture of NS4 cells can be obtained fromnormal neural tissue or, alternatively, from a host afflicted with a CNSdisease or disorder. The choice of culture conditions depends upon theparticular agent being tested and the effects one wants to achieve. Oncethe cells are obtained from the desired donor tissue, they areproliferated in vitro in the presence of a proliferation-inducing growthfactor.

The ability of various biological agents to increase, decrease or modifyin some other way the number and nature of the NS4 cells can be screenedon cells proliferated in the presence of EGF or otherproliferation-inducing factor by the methods described in EXAMPLE 1-2.

It is possible to screen for biological agents that increase theproliferative ability of NS4 cells which would be useful for generatinglarge numbers of cells for transplantation purposes. It is also possibleto screen for biological agents that inhibit NS4 cell proliferation. NS4cells are plated in the presence of the biological factors of interestand assayed for the degree of proliferation that occurs. The effects ofa biological agent or combination of biological agents on thedifferentiation and survival of NS4 cells and their progeny can bedetermined.

It is possible to screen NS4 cells which have already been induced todifferentiate prior to the screening. It is also possible to determinethe effects of the biological agents on the differentiation process byapplying them to NS4 cells prior to differentiation. Generally, thebiological agent can be solubilized and added to the culture medium atvarying concentrations to determine the effect of the agent at eachdose. The culture medium may be replenished with the biological agentevery couple of days in amounts so as to keep the concentration of theagent somewhat constant.

Changes in proliferation are observed by an increase or decrease in thenumber of neurospheres that form or an increase or decrease in the sizeof the neurospheres (which is a reflection of the rate of proliferationas determined by the numbers of NS4 cells per neurosphere). A“regulatory factor” is a biological factor that has a regulatory effecton the proliferation of NS4 cells. For example, a biological factorwould be considered a “regulatory factor” if it increases or decreasesthe number of NS4 cells that proliferate in vitro in response to aproliferation-inducing growth factor (such as EGF). Alternatively, thenumber of NS4 cells that respond to proliferation-inducing factors mayremain the same, but addition of the regulatory factor affects the rateat which the NS4 cells proliferate. A proliferation-inducing growthfactor may act as a regulatory factor when used in combination withanother proliferation-inducing growth factor.

Other regulatory factors include heparan sulfate, TGF, activin, BMP-2,CNTF, retinoic acid, TNF, MIP1, MIP-2, NGF, PDGF, interleukins, and theBcl-2 gene product. Other factors having a regulatory effect on stemcell proliferation include those that interfere with the activation ofthe c-fos pathway (an intermediate early gene, known to be activated byEGF), including phorbol 12 myristate 13-acetate (PMA; Sigma), whichup-regulates the c-fos pathway and staurosporine (Research BiochemicalInternational) and CGP-41251 (Ciba-Geigy), which down regulate c-fosexpression and factors, such as tyrphostin (Fallon et al., 11(5) Mol.Cell Biol. 2697-2703 (1991)) and the like, which suppress tyrosinekinase activation induced by the binding of EGF to its receptor.

The regulatory factors are added to the culture medium at aconcentration in the range of about 10 pg/ml to 500 ng/ml (preferably,for example, about 1 ng/ml to 100 ng/ml, or more preferably about 10ng/ml). The regulatory factor retinoic acid is prepared from a 1 mMstock solution and used at a final concentration between about 0.01 μMand 100 μM (preferably, for example, between about 0.05 μM to 5 μM).

The glycosaminoglycan, heparan sulfate, is a ubiquitous component on thesurface of mammalian cells known to affect a variety of cellularprocesses, and which binds to growth factor molecules such as FGF andamphiregulin, thereby promoting the binding of these molecules to theirreceptors on the surfaces of cells. Heparan sulfate can be added to theculture medium in combination with other biological factors, at aconcentration of about 1 ng/ml to 1 mg/ml (preferably, for example,about 0.2 μg/ml to 20 μg/ml, or more preferably about 2 g/ml).

Using these screening methods, one of skill in the art can screen forpotential drug side-effects on pre-natal and post-natal CNS cells bytesting for the effects of the biological agents on neural cellproliferation and differentiation or the survival and function ofdifferentiated CNS cells. The proliferated NS4 cells are typicallyplated at a density of about 5-10×10⁶ cells/ml. If it is desired to testthe effect of the biological agent on a particular differentiated celltype or a given make-up of cells, the ratio of neurons to glial cellsobtained after differentiation can be manipulated by separating thedifferent types of cells. Astrocytes can be panned out after a bindingprocedure using the RAN 2 antibody (available from ATCC). Tetanus toxin(available from Boerhinger Ingelheim) can be used to select out neurons.By varying the trophic factors added to the culture medium used duringdifferentiation it is possible to intentionally alter the phenotyperatios. Such tropliic factors include EGF, FGF, BDNF, CNTF, TGF, GDNF,and the like. For example, FGF increases the ratio of neurons, and CNTFincreases the ratio of oligodendrocytes. Growing the cultures on beds ofglial cells obtained from different CNS regions can also affect thecourse of differentiation.

The effects of the biological agents are identified based uponsignificant differences relative to control cultures with respect tocriteria such as the ratios of expressed phenotypes (neurons, glialcells, or neurotransmitters or other markers), cell viability andalterations in gene expression. Physical characteristics of the cellscan be analyzed by observing cell and neurite morphology and growth withmicroscopy. The induction of expression of new or increased levels ofproteins such as enzymes, receptors and other cell surface molecules, orof neurotransmitters, amino acids, neuropeptides and biogenic amines canbe analyzed with any technique known in the art which can identify thealteration of the level of such molecules. These techniques includeimmunohistochemistry using antibodies against such molecules, orbiochemical analysis. Such biochemical analysis includes protein assays,enzymatic assays, receptor binding assays, enzyme-linked immunosorbantassays (ELISA), electrophoretic analysis, analysis with high performanceliquid chromatography (HPLC), Western blots, and radioimmune assays(RIA). Nucleic acid analysis such as Northern blots and PCR can be usedto examine the levels of mRNA coding for these molecules, or for enzymeswhich synthesize these molecules.

The factors involved in the proliferation of NS4 and the proliferation,differentiation and survival of NS4 cell progeny, and their responses tobiological agents can be isolated by constructing cDNA libraries fromNS4 cells or NS4 cell progeny at different stages of their developmentusing techniques known in the art. The libraries from cells at onedevelopmental stage are compared with those of cells at different stagesof development to determine the sequence of gene expression duringdevelopment and to reveal the effects of various biological agents or toreveal new biological agents that alter gene expression in CNS cells.When the libraries are prepared from dysfunctional tissue, geneticfactors may be identified that play a role in the cause of dysfunctionby comparing the libraries from the dysfunctional tissue with those fromnormal tissue. This information can be used in the design of therapiesto treat the disorders. Additionally, probes can be identified for usein the diagnosis of various genetic disorders or for use in identifyingneural cells at a particular stage in development.

Electrophysiological analysis can be used to determine the effects ofbiological agents on neuronal characteristics such as resting membranepotential, evoked potentials, direction and ionic nature of current flowand the dynamics of ion channels. These measurements can be made usingany technique known in the art, including extracellular single unitvoltage recording, intracellular voltage recording, voltage clamping andpatch clamping. Voltage sensitive dyes and ion sensitive electrodes mayalso be used.

The details of one or more embodiments of the invention are set forth inthe accompanying description above. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are now described. Other features, objects, and advantages ofthe invention will be apparent from the description and from the claims.In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. All patents and publications citedin this specification are incorporated by reference.

The following EXAMPLES are presented to more fully illustrate thepreferred embodiments of the invention. These EXAMPLES should in no waybe construed as limiting the scope of the invention, as defined by theappended claims.

EXAMPLE 1 Dissociation of Murine Embryonic Neural Tissue andProliferation of Murine NS4 Cells

Separate embryonic (E12-15) primary neural cultures were established bymechanical or enzymatic dissociation from the striatal anlage (lateralganglionic eminence and medial ganglionic eminence) and grown in DMEM,10% FCS, N2 supplement and EGF (20 ng/ml) for 4-20 passages. The cellsgrew adherently and phenotypes were analyzed using morphology andimmunocytochemistry. For immunocytochemistry analysis, cells were fixedin 4% paraformaldehyde for 10 min. and exposed to primary and secondaryantibodies according to well-established protocols. For neuronaldifferentiation, cells were switched to serum-free medium without EGF.After 1-7 days, cells were fixed and evaluated by morphology andimmunocytochemistry. To evaluate the paternal origin of thedifferentiated progeny, cultures were established from embryonictransgenic mouse lateral ganglionic eminence and MGE expressing thereceptor of the avian RCAS virus (called tv-a) under the control of theGFAP promoter. Thus, only cells that express GFAP can be infected by anRCAS-EGFP vector, which in turn marks the cells with green fluorescence.Therefore, any neuron that is derived from a cell that once expressedGFAP is rendered fluorescent.

After a couple of passages, the parental culture shows >95% GFAP andnestin immunoreactivity. In addition, the cultures express the radialglial marker RC2. After switching to serum-free medium and removing theEGF (differentiating condition), the cells change morphology andstaining pattern to become as much as 36% neurons (as determined bymorphology and, beta-tubulin III). This finding could be reproduced withcells at least 15 passages old. The fact that the neurons had arisenfrom GFAP⁺ parental cells was established beyond doubt by using thetransgenic mouse cultures. Many of the, beta-tubulin III immunoreactivecells also expressed EGFP indicating that they had been GFAP⁺ at anearlier time point. Last, some cells retained their molecular identityand express transcription factors typical of differentiating neurons inthe lateral ganglionic eminence (e.g. DLX and MEIS2), showing that thecells are specified progenitors.

EXAMPLE 2 Dissociation of Human Embryonic Neural Tissue andProliferation of Human NS4 Cells

Human first trimester CNS tissue was collected and the LGE and MGE weredissected out and mechanically dissociated and cultured in DMEM, N2supplement; 10% FCS (“NS4 Complete Medium”), and EGF (20 ng/ml) or EGEand bFGF (20 ng/ml each). The tissues were incubated in 0.1% trypsin and0.05% DNase in DMEM for 15-20 min at 37° C. Tissue was mechanicallydissociated with a fire polished Pasteur pipette. Dissociated cells wereplated at high density in tissue culture treated flasks withoutadditional coating in “NS4 Complete Medium” and either EGF (20 ng/ml) orEGF and bFGF (20 ng/ml each). Cell cultures were housed in an incubatorat 37° C., 100% humidity, 95% air/5% CO₂. When the cultures wereconfluent, they were passaged 1:3.

After several passages (>4), parental cells were investigated for theirmorphology and expression of GFAP, nestin, and-, beta-tubulin III.Similar to the mouse cultures (EXAMPLE 1), human cells were placed inserum-free medium without growth factors and the differentiation studiedwith morphology and immunocytochemistry. Adherent human cell culturescould be established in a similar manner to mouse cultures (EXAMPLE 1).

After several passages, a majority of cells showed GFAP and nestinimmunoreactivity and glial morphology. Both the EGF and EGF and bFGFstimulated cultures appeared similar in morphology and immunoreactivepattern. Upon switching to SFM and removal of growth factors, cellsconvened into a neuronal morphology in similar or possible even largernumbers than the mouse cultures and became immunoreactive to theneuronal marker, beta-tubulin III.

EXAMPLE 3 Glial Methods

Lateral ganglionic eminence and medial ganglionic eminence sections weredissected from E13.5 or E15.5 embryos. The tissue pieces were incubatedin 0.1% trypsin and 0.05% DNase in DMEM for 15-20 min at 37° C. beforemechanical dissociation and plating, at high density in tissue culturetreated flasks without additional coating. Cells were expanded in DMEMF12 with N2 supplement (Gibco), glutamine (2 mM), antibiotics, 10% fetalcalf serum (FCS), and EGF (20 ng/ml). When the cultures were confluent,they were passaged 1:3. Both neurons and glia were present in theinitial cultures. However, by the 4th passage (P4), or after freezingand thawing, the cultures were devoid of cells possessing neuronalmorphologies or expressing neuronal markers (i.e., beta-tubulin III).These cultures were highly enriched in cells expressing nestin as wellas glial phenotypes (i.e. GFAP and RC2). In the case of LGE glialcultures, the cells were expanded extensively (passaged >25 times).These cultures expressed similar phenotypes to those passaged fewertimes.

To generate neurones a medium-switch was performed on confluent cultures(3 days after plating and splitting) from the expansion medium to thesame medium minus the serum and EGF. In some cases, a sequential switchwas performed, where first serum was removed and then EGF a few dayslater. These cultures were kept in the serum-free medium (without EGF)for 4-8 days before fixation in 4% PFA and immunostaining for neuronaland glial markers (e.g., beta-tubulin III, GFAP, nestin and RC2).

EXAMPLE 4 Longterm EGF-Stimulated Cultures of Attached GFAP⁺ Cell

In this EXAMPLE, cultures of dissociated cells prepared from lateralganglionic eminence of the mouse embryonic day 15-17 (E15-17) forebrainwere established in a medium including epidermal growth factor (EGF) andserum, to obtain propagating attached cultures with a high content ofastroglia-like cells. This EXAMPLE is to determine the long-termcharacteristics of cells cultured under these conditions. The cultureswere passaged at confluency, and growth rate, morphology and phenotypicproperties (e.g. GFAP immunoreactivity) were assessed after thesubsequent passages. The cultured cells had the morphology of astroglialcells, with the vast majority of the cells immunoreactive for GFAP(around 90%), as well as for the intermediate filament marker nestin.The cells were also positive for the mouse-specific neural antibodies M2and M6. The cells were negative when stained for the neuronal marker,beta-tubulin III.

Dissociation. Lateral ganglionic eminence tissue was retrieved mainlyfrom E15, but in a few cases also from E16-17, mouse embryos of timedpregnant mice. With the embryos immediately placed in a 1:1 mixture ofDulbecco's minimum essential medium (DMEM) and F12 (Gibco), the brainswere removed, the cortex unfolded after a medial parasagittal cut andthe underlying lateral ganglionic eminence dissected out bilaterally,using the method of Olsson et al., 69 Neuroscience 1169-82 (1995). Thetissue pieces, collected from one litter of embryos at a time, were thenplaced in a 0.1% trypsin (Worthington Biochemical Corporation)/0.05%DNase (Sigma) solution in DMEM/F12 and incubated for 20 min at 37° C.Following rinses in DMEM/F12 with 0.05% DNase, the pieces weremechanically dissociated by repeated gentle trituration through the tipsof two Eppendorf pipettes with decreasing diameters and centrifuged for5 mm at 600 rpm. The pellet was then resuspended and plated ontouncoated T75 flasks (Falcon), with a medium containing DMEM/F12supplemented with 10% Fetal Bovine Serum (FBS; Sigma), EGF (20 ng/ml,human recombinant; R & D Systems), a defined hormone and salt mixtureincluding 20 μg/nd insulin, 100 μg/ml transferrin, 20 μM progesterone,60 μM putresciene and 30 nM sodium selenite (all from Sigma Chemicals,St. Louis Colo.; see, Weiss et al., 16 J. Neurosci. 7599-609 (1996)) and1% AAS (antibiotic antimycotic solution; Sigma). The cultures weremaintained at 37° C. with 95% air and 5% CO₂, with the medium changedevery 2-3 days, and the cells passaged (or frozen down using DMSO andserum) at confluency.

In vitro cell cultures. The cultures were passaged at confluency, andgrowth rate, morphology and phenotypic properties (e.g. GFAPimmunoreactivity) were assessed after the subsequent passages. After aninitial in vitro study, with the cells evaluated over five passages (“Invitro A”), a second more extensive in vitro characterization (“In vitroB”) was initiated. To address the importance of the EGF, controlcultures without the trophic factor were studied both during the initialculture period, and at later passages.

In the initial in vitro cultures (“In vitro A”), E15-17 LGE-cells fromthree litters were dissociated, split and plated into six T75s, andsubsequently split 1:5 at confluency into T75s (n=6), with assessment ofgrowth rate and proportion of GFAP-immunoreactive over the first fivepassages.

In the second more extensive and long-term cultures (“In vitro B”), LGEtissue was obtained from three E15 litters, with plating into two T75flasks for each litter. Thus, six different cultures were followed overthe subsequent passages. When the cells reached confluency, a definednumber of 1.7×10⁶ cells were plated onto each new T75.

After each of the passages, the cell doubling time (t₂) and days neededto reach confluency were assessed and the mean values for the sixdifferent cultures plotted as functions of the passage number. t₂ wasdefined as the natural logarithm of 2 divided by the k-value (1n2/k) ofa growth curve showing the change in cell number (logarithmized) as afunction of time. The growth curve was determined by placing a countinggrid at six randomly marked positions at the bottom of each flask, andcalculating the average cell number in the grid area for each flask(n=6) and time-point. Initially, the growth curves were based on cellcounts made 1-2 times per day, as compared to only 2-3 times a weekduring the more slowly proliferating stages.

As shown in FIG. 5, which presents data from the second more extensiveculture cultures (“In vitro B”), measurements of “Days to confluency”and “Cell doubling time” revealed a high mitotic activity in thecultures after each of the first four passages, but with a gradualdecrease in growth rate, which continued and became more significantafter passages 6-8. The mean of the “Cell doubling time” during passages5-8 was significantly higher than during the initial four passages(paired Student t test, p<0.01). The same growth pattern was alsoobserved in the first set of cultures (“In vitro A”), although then onlyquantified over the first five passages (data not shown), but when thecultures were left to grow further for several months, it was found thatafter a period of slow growth, the cells started to divide more readilyagain. Similarly, in the second set of cultures (“In vitro B”), the rateof division increased again after passage nine and thereafter stabilizedat values equal to the initial culture period (FIG. 5).

Observations of in vitro cell cultures. From a few days after thedissection and initial plating, the cultures grew well, with smallerphase-bright cells and clusters of tightly aggregated cells, resemblingso-called “neurospheres” (Reynolds & Weiss, 175 Dev. Biol. 1-13 (1996)),situated on top of dense islands of attached cells. Already afterpassage two, the cultures were more homogeneous, with small and tightislands of elongated attached cells, with short arm-like processes. Overthe first few passages, the majority of the cells gradually became moreflat and epitheloid, with a cubical or polygonal shape, and with fewerprocesses and more round and distinct nuclei, and thus resembling type Iastroglial cells. During the slowly dividing passages (passages 5-8) themorphology of the cells changed, to a larger and more flat and roundappearance, with long and thin processes and with cells less denselyaggregated on the bottom of the flask. After passage 8 the cellsreassumed the morphology of the early passages.

In control cultures from passage 11 without EGF, cells attached wellafter plating, but ceased to proliferate further, as followed over fiveweeks. The cell morphology also changed into a larger and more flatappearance, with long and thin processes extending from the cell body.Separate cultures were also prepared, without EGF already from the firstplating and start of the cultures, and here no or very little growth wasobserved during the subsequent six weeks. These cells had a morphologyresembling that of the passage 11 control cultures after EGF removal.

Immunoreactivity. GFAP-immunocytochemistry, performed after each of thefirst five passages and after passage 18, revealed that around 75% ofthe cells were GFAP⁺ already after passage two, and with approximately90% GFAP⁺-immunoreactive cells after passage five, and also afterpassage 18 (FIG. 4). A similar proportions of the cells also expressedthe intermediate filament nestin, both at the early and late passages.The mouse-specific neural markers M2 and M6 were also detected in themajority of the cells, overlapping with the GFAP and nestinimmunoreactivities, but with a reduced expression of M2 at the laterpassages, and with M6 in general expressed at lower levels than M2. Noor only occasionally, beta-tubulin III⁺ cells were detected at eitherpassage five or 18. For immunocytochemistry, 100,000 cells were platedin uncoated 4-well plates (NUNC) after each passage, and afterattachment fixed in 4% paraformaldehyde (PFA). After rinses withpotassium phosphate buffered saline (KPBS), the cultures werepreincubated with 5% normal serum raised in the same species as thesecondary antibody, in 0.02 M KPBS for 1 hr at room temperature (RT).Following incubation with primary antibodies (overnight at 4° C.), thecultures were rinsed three times in 0.02 M KPBS (with 5% serum), andincubated with a biotinylated secondary antibody (2 hrs, 1(T), rinsed inKPBS and incubated with an avidin-biotin-peroxidase complex(Vectastain-Elite ABC Kit PK-6 100) using 3,3-diaminobenzidine aschromogen (25 mg/ml; Sigma).

Analysis of long-term attached cultures of GFAP⁺ nestin⁺ cells. Weobserved poor plating and growth in the control cultures initiatedwithout EGF. We also observed a shift in growth rate during theextensive culture period, with about 25 passages over 7 months. Theinitially high rate of cell division gradually decreased over the first8 passages, after which the cells increased their growth rate again,stabilizing at values equal to the first 4 passages, thus with featuresof a cell line (FIG. 5).

The early and late passage cells have many similarities in addition tothe morphological and immunocytochemical characteristics presented here,such as immunoreactivity also for the radial glia marker RC-2 (FIG. 3).

EXAMPLE 5 Differentiation of NS4 Cells into Neurons and Glia

To generate neurons, cells were proliferated as in EXAMPLES 1 or 2 onuncoated tissue culture plastic. Three days after plating, a mediumswitch was performed from the expansion medium to the same medium minusthe serum and EGF. In some cases, a sequential switch was performed,where first serum was removed and then EGF a few days later. Culturesfor indirect immunocytochemistry, were kept in the serum-free medium(without EGF) for 4-8 days before fixation in 4% paraformaldehyde inPBS. Following a 10 min fixation, coverslips were washed three times inPBS and immunostained for neuronal and glial markers (e.g.beta-tubulinIII, GFAP, nestin, and RC2). Coverslips were incubated with primaryantiserum in PBS/10% normal goat serum, 0.3% TRITON-X-100 for two hoursat 37° C. Coverslips were washed 3× in PBS and incubated with labeledsecondary antibodies for 30 mm at 37° C. Coverslips were then washed 3×in PBS, rinsed with water and placed on glass slides. Between 17-36% ofthe cells derived from cell cultures established from differentdissections including human LGE, were neurons as determined bymorphology and beta-tubulin III staining. To further examine theneuronal differentiation, ICC staining for GABA and calbindin were doneindicating that most of the cells formed a GABA-ergic neuronalphenotype. Furthermore, the cells showed other evidence of retention ofthe striatal specification such as the expression of the marker DLX1 andMEIS2 but not PAX6 and NKX2.1, markers of cortical and MGE neuronsrespectively. The neuronal phenotype and function was further confirmedby electrophysiology which demonstrated electrochemical activitycharacteristic of neurons. During differentiation, many cells co-labeledwith both beta-tubulin III and GFAP. With further differentiation matureneuronal and astrocytic phenotypes and separate beta-tubulin III andGFAP immunoreactivity were observed.

EXAMPLE 6 Generation of Neurons from Multipassage Glial Cultures andConfirmation of Glial Origin

Embryonic glial cells can be grown and expanded for several months inEGF and serum-containing medium, with a majority of the cells expressinghigh levels of GFAP and nestin, even after 25 passages. In this EXAMPLE,we used such glial cultures derived from the mouse telencephalon (E13.5and E15.5); the LGE (lateral ganglionic eminence) and the MGE (medialganglionic eminence). We show that after 4 to 25 passages, high numbersof neurons can be generated from these cells simply by removing serumand EGF from their culture medium.

The neurons we generate turn on, not only the neuron-specific marker-,beta-tubulin III, but also MEIS2 and DLX, transcription factors specificfor the regions where the glia were dissected from.

To determine whether the neurons actually derive from glial cells weused cell cultures from the GFAP-tva mouse. These mice express thereceptor for the RCAS-virus, tva, under the GFAP promoter (see, EXAMPLE1). Thus, only GFAP expressing cells can be infected. After infectionwith an RCAS(A)GFP (green fluorescent protein) virus, GFP cells werefound that were also, beta-tubulin III⁺ and had a neuronal morphology.These results show that a subpopulation of GFAP⁺ cells in multipassageglial cultures derived from the ventral telencephalon are indeedneuronal precursors.

EXAMPLE 7 Transplantation of NS4 Cells into Adult and Neonatal Rats

After passages 4 to 6 and passage 18 of the cells in EXAMPLE 3, in vivostudies were conducted to analyze the survival, morphology and migratorypatterns of the cultured cells after transplantation. Thus, cellsuspensions were prepared from the cultures, and implanted cross-speciesinto the striatum of immunosuppressed intact or lesioned adult rats, andinto neonatal rats. At 4 weeks post-transplantation, the grafts wereanalyzed through immunohistochemistry and in situ hybridization, usingspecies-specific and phenotypic markers.

Method of transplantation into adults. Twelve adult femaleSprague-Dawley rats (BK Universal, Sweden) were the adult graftrecipients. For surgery, the rats were anaesthetized with Equithesin(0.3 ml/100 g body weight) and placed in a Kopf stereotaxic frame. Atotal of 1.4 l ibotenic acid (10 g/l dissolved in 0.1M phosphate buffer(PB; Sigma) were injected into the head of the right caudate-putamen,divided over three sites:A=+0.2; L=−3.0; V=−5.5 (0.5 l);  (1)A=+0.2; L=−3.0; V=−4.0 (0.5 ll);  (2)A=+1.5; L=−2.5; V=−4.7 (0.4 pl)  (3)

(tooth bar at 2.3; the ventral (V) coordinate was measured from thedural surface; A, anterior and L, lateral to bregma).

One to two weeks after the lesion, suspensions with cells from passages4-5 cultures were implanted into the ibotenic acid-lesioned area andinto the contralateral intact striatum of the adult recipient rats. Toprepare the cell suspensions, the culture medium was replaced by Hank'sBalanced Salt Solution without magnesium and calcium (HBSS; GIECO), for1 min and after an incubation with 0.1% trypsin solution for 4 mm at 37°C., serum-containing medium was added and the cells were detached fromthe flasks. After centrifugation for 5 mm at 600 rpm, the cells wereresuspended in HBSS (with magnesium and calcium), counted in ahemocytometer, and prepared into a single cell suspension with a celldensity of 50,000 cells/. The suspensions were injected from a Hamiltonsyringe with a total of 2 or 4 pl implanted into each striatum at thefollowing coordinates:A=+0.4; L=+/−2.8; V=(a) −5.0, (b) −4.5;  (1)A=+0.8; L=+/−2.5; V=(a) −5.0, (b) −4.5  (2)

(tooth bar at 2.3).

The adult host rats were immunosuppressed by daily intraperitoneal (ip)injections of cyclosporin A (10 mg/mm; 1 ml/kg body wt), from one daybefore transplantation until sacrifice.

Method of transplantation into neonates. Cell suspensions from passages4-6 (“Neonate a”) and passage 18 (“Neonate b”) cultures (from EXAMPLE 4)were injected into the striatum bilaterally in 27 neonatal rats andunilaterally into five neonates, with a cell density adjusted to 25 000cells/p1 or 100,000 cells/pl. The injections were made from glasscapillaries attached to a Hamilton syringe and in all cases the neonateswere placed in a neonatal frame during surgery (see, Cunningham & McKay,47 J. Neurosci. Methods 105-14 (1993)). In total, 2 1/neonatal striatumwere injected at the following coordinates:A+0.5; L+/−2.2; V (a) −3.0, (b) −2.5;  (1)A+0.9; L+/−1.9; V (a) −3.0, (b) −2.5;  (2)

with bregma and lambda at the same horizontal level.

Immunohistochemistry after transplantation. Four weeks aftertransplantation, the rats were anaesthetized with an overdose ofpentobarbital (ip) and transcardially perfused with 200-300 ml ice-cold4% PFA in 0.1 M phosphate buffer (PB). The brains were removed,postfixed for 6 hrs in the same fixative and then soaked overnight in0.1 M PB containing 25% sucrose. The brains were sectioned in thecoronal or sagittal planes at 30 m, using a sliding knife freezingmicrotome (Leica SM 20001). Endogenous peroxidase activity was quenchedwith 3% H₂O₂ in KPBS (10 min). The sections were reacted as describedabove for the in vitro immunocytochemistry, using either biotinylated,or Cy2-conjugated or Cy3-conjugated secondary antibodies. The sectionswere then mounted onto chrome-alum coated slides, dehydrated andcoverslipped with DPX (BDH). The sections incubated with aCy2-conjugated or Cy3-conjugated secondary antibody were directlymounted and coverslipped with PVA/DABCO.

In selected sections, double immunohistochemistry was performed tosimultaneously visualize both M2 and M6 or both M2 and GFAP. For the M6and M2, double staining was performed. The M6 epitope was first labeledwith the M6 antiserum and a secondary Cy3-conjugated anti-rat antibody,followed by M2-staining with a primary biotinylated M2-antiserum and anavidin-Cy2-complex. For the different protocols, controls with omissionof primary antibodies were negative.

In situ hybridization. Sections were processed for in situ hybridizationto detect mouse satellite DNA. After additional fixation for 10 min in4% PFA, the sections were first incubated in 2×SSC and 5 mM EDTA at 37°C., and then digested with protease from Streptomyceus griseus (25 g/ml;Sigma) in 2×SSC and 5 mM EDTA (pH 8.0) for 10 min at 37° C. Thenfollowed dehydration in ascending ethanols, and denaturation in 70%formamide (2×SSC) for 10 min at 90° C. After additional dehydration withice-cold ethanols, hybridization was carried out overnight at 37° C.with a digoxigenin end-labeled oligonucleotide probe to mouse satelliteDNA, in 65% formamide, 2×SSC, 250 g salmon sperm DNA. After washes, thehybridized probe was visualized using a fluorescein-labeled antibody todigoxigenin (Boehringer Ingelheim). After in situ hybridization, thesections were additionally stained with antibodies against either GFAPor NeuN, using the same protocol as described above, although performedon these already mounted sections, and with a Cy3-labeled secondaryantiserum.

In vivo/transplantation studies. TABLE 1 summarizes the outcome of thetransplantations, with cells from passages 5-6 implanted into adultintact and lesioned striatum, and cells from either passages 4-6 orpassage 18 injected into the neonatal striatum (“Neonate a” and “Neonateb”, respectively).

TABLE 1 Summary of the grafts implanted into the striatum of intact orlesioned adult rats, and of neonatal rats. The neonatal rats receivedcells from either passages 4-6 (“Neonate a”) or passage 18 (“Neonateb”), and the adult animals from passage 5-6. Note the differences in theamounts of cells transplanted per striatum. Donor Number Number ofTransplant cells of cells/ Number of positive group Treatment (Passage)striatum transplants transplants Adult Lesion 5 200,000 7 4 Intact 5200,000 7 5 Lesion 4 100,000 5 5 Intact 4 100,000 5 5 Total 24 19“Neonate a” Intact 6 100,000 14 5 Intact 6 200,000 5 1 Intact 4 100,00014 10 Intact 6 50,000 10 5 Total 43 21 “Neonate b” Intact 18 100,000 1611 Total 16 11

In the adult transplanted animals, surviving cells were found in 9 outof 11 lesioned and 10 out of 12 intact striata (TABLE 1). As shown inTABLE 2, the M2⁺ cells were primarily located along the needle tract,densely aggregated in a graft core and with only a restricted migrationinto the surrounding striatum, both after implantation into the intactand into the lesioned side. The graft core was significantly larger inthe specimens receiving 200,000 as compared to 100,000 cells(F(1.15)=6.7, p<0.05, two-way ANOVA), but not significantly differentbetween the lesioned and intact sides, although there was a trendtowards larger grafts and graft cores on the lesioned sides (TABLE 2).Migration of M2⁺ cells occurred for up to 0.6 mm away from the needletract, with no significant effect of either the implantation site(intact vs. lesion) or number of implanted cells (TABLE 2). Cells in theperiphery of the core, and cells that had migrated for some distance,presented an astroglia-like morphology with many short processes.

TABLE 2 Summary of the adult recipients which containedM2-immunoreactive grafts, with measurements of the diameter of the graftcore (m), the maximal cell migration distance (m) (intact and lesionedside respectively) and with the mean numbers for each of the groups withdifferent numbers of implanted cells. The graft core was defined as theregion of dense M2-immunoreactivity around the needle tract and themaximal cellmigration distance was measured from the visible needletract. Intact side Lesioned side Maximal Maximal Number of Diameter ofcell Diameter cell Animal transplanted graft core migration of graftmigration No. cells/striatum (Om) (Om) core (Om) (Om) 1 200,000 350 210950 710 2 850 500 800 650 3 260 950 — — 4 400 1050  400 300 5 400 500460 350 Mean 452 642   652.5   502.5 6 100,000 350 250 400 550 7 250 500550 500 8 120 500 200 100 9 250 500 300 340 10  200 600 600 400 Mean 234470 410 378

When grafting cells from passages 4-6 into neonates (“Neonate a”), M2⁺grafts were found in 21 out of 43 grafted striata (TABLE 1), with nomarked differences in graft survival or migration patterns due todifferences in the numbers of implanted cells (TABLE 3). Thetransplanted cells migrated all over the striatum, along the internalcapsule and in large numbers into the globus pallidus, with smallernumbers of cells distributed also in other adjacent areas (TABLE 3). TheM2⁺ cells were small and bush-like with numerous short processes andappeared morphologically similar to mature gray matter (type I)astrocytes.

With grafts of passage 18 (i.e. six month-old) cultures, M2⁺ cells werefound in 11 out of 16 grafted striata (TABLE 1). As shown in TABLE 3,these implants were similar to the early passage grafts, both incellular morphology and migration patterns, although with an overallreduction in the total number of M2⁺ cells.

TABLE 3 Summaries of the neonatal recipients containingM2-immunoreactive grafts after implantation of cells from (a) passages4-6 (“Neonate a”) or (b) passage 18 (“Neonate b”). The TABLE shows thenumbers of cells transplanted per striatum, the distribution and arelative rating of the numbers of M2⁺ cells integrated into differentregions of the recipient brain (+++, high numbers; ++, moderate numbers;+, low numbers; - no cells detected). cc, corpus callosum; cx, cortex;EP, entopeduncular nucleus; Fr, frontal cortex; hpc, hippocampus; ic,interna capsula; s, septum; Sfi, septofimbrial nucleus; sin, striamedullaris thalamus; Th, Thalamus, VP, Ventral Pallidum. Number ofAnimal transplanted Globus Single cells no. cells/striatum StriatumPallidus in other areas “Neonate a” (cells from passages 4-6). 1 Right100,000 +++++ + Cx, cc Left 100,000 ++++ + — 2 Right 100,000 — — — Left100,000 ++ + — 3 Right 100,000 +++ +++++ Cc, cx, ic, septum, Sfi Left100,000 +++ +++++ cc, hpc, septum, Sfi 4 Right 200,000 ++++ +++ c, cx,ic, septum 5 Right 100,000 ++++ ++++ Septum, Sfi Left 100,000 ++++ ++++septum, Th 6 Right 100,000 +++++ ++++ Cx, EP, hpc, ic, SFi, Th Left100,000 +++++ +++++ hpc, ic, SFi, VP 7 Right 100,000 ++ ++ — Left100,000 +++ ++ — 8 Right 100,000 ++++ ++ ic, Left 100,000 ++++ +++ cx,ic 9 Right 100,000 +++ +++ Cc, hpc, ic, SFi, hpc Left 100,000 +++ ++hpc, ix, SFi, sm 10 Right 50,000 — ++ Cc, ic Left 50,000 — ++ ic 11Right 50,000 ++ + Cc, ic Left 50,000 ++ ++ cc, Fr 12 Right 50,000 + ++Cc, cx Left 50,000 — — — “Neonate b” (cells from passages 18). 1 Right100,000 +++ ++++ Cc Left 100,000 +++++ +++++ Ic 2 Right 100,000 ++ ++++Ic Left 100,000 + +++ ic, hpc, VP 3 Right 100,000 +++ ++ Ic Left 100,000++++ +++ — 4 Right 100,000 ++ +++ ic, Left 100,000 ++ ++ ic, hpc, Th 5Right 100,000 +++ + Hpc Left 100,000 ++++ + Ic 6 Right 100,000 ++ — —Left 100,000 — — —

In both the neonatal and adult recipients, the majority of the M2⁺ cellswere also M6-immunoreactive. There were, however, no axon-likeprojections emanating from the regions of M2⁺/M6⁺ cells. Using DNA insitu hybridization with a probe recognizing mouse, but not rat,satellite DNA (Br,stle et al., 15 Neuron 1275-85 (1995)), it waspossible to confirm the distribution of the M2⁺ cells within both adultand neonatal recipients. The concentration of cells in the globuspallidus, as revealed by M2-staining, was evident also with thesatellite DNA-method. Simultaneous double staining for mouse satelliteDNA and with GFAP immunohistochemistry, revealed that in the globuspallidus around 75% of the mouse satellite DNA⁺ cells were also found tobe GFAP-immunoreactive. In the striatum, the overallGFAP-immunoreactivity was lower, with only around 37% of the satelliteDNA-labeled cells clearly GFAP⁺. Using a similar double labelingprotocol, only occasional single mouse satellite-stained cells (0.5%)were also immunopositive for the neuronal antigen NeuN, either in thestriatum or in the globus pallidus.

Discussion. Immunohistochemistry for M2 showed that the implanted cellsdeveloped an astroglia type I-like morphology, with a distributionoverlapping that obtained by mouse-satellite DNA in situ hybridization(Bristle et al., 15 Neuron 1275-85 (1995)). Astroglia-like M2+ cellswere also positive for M6. No axonal projections were found to emanatefrom the implanted cells, and only occasional mouse-satellite DNA⁺ cellscould be labeled also with the neuronal marker NeuN. Thus, the implantedcells can survive and integrate well, and acquire an astroglialphenotype after implantation, both when grafted into the neonates andinto the adult (lesioned and intact) recipient brains.

These findings from the grafts placed into the neonates and from theadult recipients are in agreement with previous work with grafts ofprimary ganglionic eminence tissue implanted into rat hosts of differentdevelopmental stages (Olsson et al., 79 Neuroscience 57-78 (1997)).

The astroglial nature of the implanted cells was further evidenced bythe finding that around 75% of the implanted cells (mouse-satelliteDNA⁺) were GFAP⁺ in the host globus pallidus. In the striatum, thenumber of double labeled cells was lower, but also the overallGFAP-staining of the host brain was lower in that region. Interestingly,the pattern of GFAP-immunoreactivity in the grafted cells was thusregionally similar to that of the surrounding host brain.

The migration of astroglial cells from primary tissue-grafts is regionspecific, and thus dependent on from where the tissue is dissected(Gates et al, 84 Neuroscience 10 13-23 (1998)). Questions regarding theregional specificity of astroglial cells, could also be furtheraddressed by growing relatively pure populations of astroglial cellsfrom different CNS regions using the present or a similar cultureprotocol.

Importantly, no tumors were formed when implanting late passage cells,even though the cells showed a high growth rate in vitro. Although thelate passage cells seemed to survive less well than the early passageones, also the late passage cells had a clear astroglial morphology andshowed similar migration patterns when grafted into the neonatalrecipients.

The foregoing descriptions have been presented only for the purposes ofillustration and is not intended to limit the invention to the preciseform disclosed, but by the claims appended hereto.

1. An in vitro cell culture of GFAP⁺ nestin⁺ cells, wherein a) one ormore cells in the culture have the capacity to differentiate intoneurons; b) the cell culture divides in a culture medium containing atleast one proliferation-inducing growth factor; and c) one or more cellsin the culture differentiate into neurons upon withdrawal of theproliferation-inducing growth factor.
 2. The cell culture of claim 1,wherein the cells are derived from the central nervous system of amammal.
 3. The cell culture of claim 1, wherein the cell culturedifferentiates into at least 10% neurons under differentiation-inducingculture conditions.
 4. The cell culture of claim 1, wherein, underdifferentiation-inducing culture conditions, the majority ofdifferentiated neuronal cells have a GABA-ergic phenotype.
 5. The cellculture of claim 1, wherein the culture is capable of at least 6doublings.
 6. The cell culture of claim 1, wherein the cells are derivedfrom the lateral ganglionic eminence (LGE) or medial ganglionic eminence(MGE) of a mammal.
 7. The cell culture of claim 1, wherein the doublingrate of the culture is faster than seven days.
 8. The cell culture ofclaim 1, wherein the cells in the culture are murine.
 9. The cellculture of claim 1, wherein the cells in the culture are human.
 10. Thecell culture of claim 1, wherein fewer than 5% of the cells in theculture are β-tubulin III immunoreactive (β-tubulin III⁺) underproliferation-promoting culture conditions and between 10-40% of thecells in the culture are β-tubulin III immunoreactive (β-tubulin III⁺)under differentiation-inducing culture conditions.
 11. The cell cultureof claim 1, wherein the proliferation-inducing growth factor is selectedfrom the group consisting of epidermal growth factor, amphiregulin,basic fibroblast growth factor, acidic fibroblast growth factor,transforming growth factor alpha, leukemia inhibitor factor, ciliaryneurotrophic factor and combinations thereof.
 12. The cell culture ofclaim 1, wherein one or more of the cells in culture differentiate intoglia in the absence of the proliferation-inducing growth factor from theculture medium.
 13. The cell culture of claim 12, wherein the glia areboth GFAP⁺ and vimentin positive.
 14. The cell culture of claim 12,wherein the morphology of the glia is: (a) bipolar; (b) elongated; and(c) non-fibrillary.
 15. The cell culture of claim 1, wherein one or moreof the cells in culture, under differentiation-inducing cultureconditions, differentiate into neurons that exhibit: (a) axon-dendritepolarity, (b) synaptic terminals, and (c) localization of proteinsinvolved in synaptogenesis and synaptic activity.
 16. The cell cultureof claim 15, wherein the proteins involved in synaptogenesis andsynaptic activity are (i) neurotransmitter receptors, (ii) transporters,or (iii) processing enzymes.
 17. The culture of claim 1, wherein themajority of differentiated neuronal cells are immunoreactive withstriatal neuronal markers.
 18. The culture of claim 1, wherein themajority of differentiated neuronal cells are not immunoreactive withcortical neuronal markers.
 19. The culture of claim 1, wherein themajority of differentiated neuronal cells are not immunoreactive withneuronal markers of the medial ganglionic eminence.
 20. The cell cultureof claim 1, wherein the majority of cells are immunoreactive to GFAP andnestin.
 21. The cell culture of claim 20, wherein the GFAP⁺ nestin⁺cells show glial morphology.
 22. The cell culture of claim 1, wherein atleast about 75% of cells are immunoreactive to GFAP and nestin.
 23. Thecell culture of claim 22, wherein the GFAP⁺ nestin⁺ cells show glialmorphology.
 24. The cell culture of claim 1, wherein at least about 90%of cells are immunoreactive to GFAP and nestin.
 25. The cell culture ofclaim 24, wherein the GFAP⁺ nestin⁺ cells show glia morphology.